Small mobile stem cells (sms) and uses thereof

ABSTRACT

The presently disclosed subject matter relates, in general, to the identification, isolation, and use of a population of stem cells isolated from umbilical cord blood, peripheral blood and/or other sources and that are referred to herein as Small Mobile Stem cells (short: SMS). More particularly, the presently disclosed subject matter relates to isolating said SMS stem cells and employing the same, optionally after in vitro manipulation, to treat tissue and/or organ damage in a subject in need thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 61/833,467, filed Jun. 11, 2013, which is incorporated by referenceherein in its entirety.

FIELD OF THE INVENTION

The presently disclosed subject matter relates, in general, to theidentification, isolation, and use of a population of stem cellsisolated from one or more sources, including but not limited toumbilical cord blood, peripheral blood, bone marrow, solid tissues suchas placenta, liver, heart, brain, kidney and gastro intestinal tract,and that are referred to herein as Small Mobile Stem cells (SMS). Moreparticularly, the presently disclosed subject matter relates toisolating said SMS stem cells and employing the same, optionally afterin vitro manipulation, to treat tissue and/or organ damage in a subjectin need thereof.

BACKGROUND OF THE INVENTION

The use of stem cells and stem cell derivatives is currently of greatinterest to medical research, particularly for the prospects ofproviding reagents for treating tissue damaged by various causes such asgenetic disorders, injuries, and/or disease processes. In theory, stemcells, capable of asymmetric division; replenishing them self andproviding various differentiated cell types could replace any damagedcells and tissues of an organism of choice. This process of regenerationis inherently present, to various extents, in all living multicellularorganisms. Human organs however vary greatly in their potential forregeneration and repair. Many vital organs such as the heart and thebrain show little capacity for repair after injury.

A lot of effort was concentrated at isolating and identifying human stemcells from a number of different tissues for use in regenerativemedicine. And since bone marrow transplants have been successfullyperformed for decades, such efforts were concentrated initially onidentifying stem cells in bone marrow. U.S. Pat. No. 5,750,397 disclosesthe isolation and growth of human hematopoietic stem cells that arereported to be capable of differentiating into lymphoid, erythroid, andmyelomonocytic lineages. U.S. Pat. No. 5,736,396 discloses methods forlineage-directed differentiation of isolated human mesenchymal stemcells under the influence of appropriate growth and/or differentiationfactors. The derived cells can then be introduced into a host formesenchymal tissue regeneration or repair.

Another area of interest was the use of embryonic stem (ES) cells. Thesestem cells have been shown in mice to have the potential todifferentiate into all the different cell types of the animal. Mouse EScells are derived from cells of the inner cell mass of early mouseembryos at the blastocyst stage, and other pluripotent and/or totipotentcells have been isolated from germinal tissue (e.g., primordial germcells; PGCs). Unfortunately, the development of human ES (hES) cells wasnot as successful.

In addition to the ethical controversy inherent to the use of human EScells, significant other challenges face the use of ES cells or otherpluripotent cells for regenerative therapy. The control of growth anddifferentiation of the cells into the particular cell type required fortreatment of a subject is difficult. There have been several reports ofthe effect of growth factors on the differentiation of ES cells. Forexample, Schuldiner et al. report the effects of eight growth factors onthe differentiation of cells into different cell types from hES cells(Schuldiner et al. (2000) 97 ProcNatlAcadSci USA 11307-11312). Asdisclosed therein, after initiating differentiation through embryoidbody-like formation, the cells were cultured in the presence of bFGF,TGFβ1, activin-A, BMP-4, HGF, EGF, βNGF, or retinoic acid. Each growthfactor had a unique effect on the differentiation pathway, but none ofthe growth factors directed differentiation exclusively to one celltype. Also the current strategies for isolating ES cell lines,particularly human ES cell lines, preclude isolating the cells from asubject and reintroducing them into the same subject (autologoustransfer). The use of a subject's own cells would obviate the need foradjunct immunosuppressive therapy, maintaining thereby full competencyof the immune system.

Adult human stem cells such as MSCs have been shown to have thepotential to differentiate into several lineages including bone(Haynesworth et al. (1992) 13 Bone 81-88), cartilage (Mackay et al.(1998) 4 Tissue Eng 415-28; Yoo et al. (1998) 80 J Bone Joint Surg Am1745-57), adipose tissue (Pittenger et al. (2000) 251 Curr TopMicrobiolImmunol 3-11), tendon (Young et al. (1998) 16 J Orthop Res406-13), muscle, and stroma (Caplan et al. (2001) 7 Trends Mol Med259-64).

Another population of cells, multipotent adult progenitor cells (MAPCs),has also been purified from bone marrow (BM; Reyes et al. (2001) 98Blood 2615-2625; Reyes & Verfaillie (2001) 938 Ann NY AcadSci 231-235).These cells have been shown to be capable of expansion in vitro for morethan 100 population doublings. MAPCs have also been shown to be able todifferentiate under defined culture conditions into various mesenchymalcell types (e.g., osteoblasts, chondroblasts, adipocytes, and skeletalmyoblasts), endothelium, neuroectoderm cells, and more recently, intohepatocytes (Schwartz et al. (2000) 109 J Clin Invest 1291-1302).

In vivo experiments in humans demonstrated that transplantation of CD34⁺peripheral blood (PB) stem cells led to the appearance of donor-derivedhepatocytes (Korbling et al. (2002) 346 N Engl J Med 738-746),epithelial cells (Korbling et al. (2002) 346 N Engl J Med 738-746), andneurons (Hao et al. (2003) 12 J Hematother Stem Cell Res 23-32).Additionally, human BM-derived cells have been shown to contribute tothe regeneration of infarcted myocardium (Stamm et al. (2003) 361 Lancet45-46). Currently Adult stem cells such as mesenchymal stem cells arewidely investigated in clinical trials for a variety of diseases (Ali etal. (2012) 2 (1) Stem Cell Discovery 15-23).

Recently a population of very small stem cells has been isolated usingFACS cell sorting. These were named very small embryonic like stem cell(VSEL). This is a rare cell population that possess very primitivemorphology and express pluripotent stem cell markers (e.g., Oct4, Nanog,and SSEA-4) as well as the surface phenotype Sca1+/CD133+Lin−CD 45− inmice/humans. VSELs can be mobilized into peripheral blood followingacute myocardial infarction (Kucia et al. (2008) 26 Stem Cells2083-2092), and is reported to improve heart function and alleviatecardiac remodeling (Dawn et al. (2008) 26 Stem cells 1646-55, Zuba-Surmaet al. (2011) 15 J Cell Mol Med 1319-28).

Attempts to culture these cells (VSEL) were unsuccessful, which led someresearchers to question their very presence in human (Danova et al.(2012) 7 PLoS One e34899). Other researchers such as Gu et al. (2013)found also that it is very difficult expand or culture human VSELs invitro using general culture conditions. Thus it is not clear yet whetherthese cells are merely developmental remnants found in the adult tissuethat cannot be harnessed effectively for regeneration or that theyrepresent real stem cell population suitable for regenerative medicine.

Generally obtaining Adult stem cells from tissues other than bone marrowcontinues to be difficult; especially for the case of providingsufficient cells for autologous transfer. Non autologous transfer ofcells implanting stem cells to others is on the other hand prone toproblems associated with an immune rejection reaction and would requirean adjunct immune suppressive therapy. Ex vivo culturing of adult stemcells is used as an alternative for providing sufficient cells. Howeveradult stem cells are relatively sensitive to incubation conditions andif successfully cultured require strict control of these conditions(Bhattacharya et al., (2009) Frontiers of cord blood science,springer-verlag London Limited).

The concept of transdifferentiation of adult tissue-specific stem cellsis a topic of extensive disagreement within the scientific and medicalcommunities (see e.g., Lemischka (2002) 30 ExpHematol 848-852; Holden &Vogel (2002) 296 Science 2126-2129). Studies attempting to reproduceresults suggesting transdifferentiation with neural stem cells have beenunsuccessful (Castro et al. (2002) 297 Science 1299). It has also beenshown that the hematopoietic stem/progenitor cells (HSPC) found inmuscle tissue originate in the BM (McKinney-Freeman et al. (2002) 99ProcNatlAcadSci USA 1341-1346; Geiger et al. 100 Blood 721-723; Kawada &Ogawa (2001) 98 Blood 2008-2013). Additionally, studies with chimericanimals involving the transplantation of single HPCs into lethallyirradiated mice demonstrated that transdifferentiation and/or plasticityof circulating HPSC and/or their progeny, if it occurs at all, is anextremely rare event (Wagers et al. (2002) 297 Science 2256-2259).

The clinical experimental use of stem cells is based mainly on providingindividual cells that are more or less differentiated. The success ofengraftment is dependent on introduced cells homing to the correctlocation and positioning there in a correct manner that would create afunctional tissue (Chute et al (2006) 13(6) CurrOpinHematol. 399-406).Reliance on cell therapy, as opposed to tissue therapy, is mainly due tothe difficulty of inducing cells to structure tissue typicalmacrostructures in vitro. Many of the newly investigated and introducedinnovations are targeting this problem by applying natural or artificialscaffolds or various other means, including the use of extracellularmatrix derived from other cells and tissues.

There continues to be a need in cell therapy for new approaches togenerate populations of transplantable cells suitable for a variety ofapplications. These cells should be easy to isolate, cheap to maintain,and provide efficient ex vivo proliferation and differentiation. On theother hand there is the need for stem cells capable of establishing invitro pre-prepared tissue like structures, engineered preferably ofautologous components.

SUMMARY

Disclosed herein, in certain embodiments, is a newly isolated humanadult stem cell: Small Mobile Stem cell (SMS). In various embodiments,this cell line is isolated from umbilical cord blood, peripheral blood,bone marrow, solid tissues such as placenta, liver, heart, brain,kidney, or gastro intestinal tract.

The SMS cells described herein are technically stem cells (i.e.,undifferentiated cells of a multicellular organism that are capable ofgiving rise to indefinitely more cells of the same type, and from whichcertain other kinds of cells arise by differentiation) but havesignificant difference. Specifically, SMS cells do not merely regeneratelost cells but rather have the ability to reconstruct a whole complexset up of tissue-like structures in the absence of special stimuli orsupporting agents.

In specific embodiments, the SMS cells are successfully cultured for atleast 3 months using the same growth medium without a serum, e.g., FetalBovine Serum (FBS). In these embodiments, the cells are cultured withoutserum with no significant effect on the proliferation potential, shapeor appearance of the cells.

In contrast to cells which affect the positioning of other cells byremotely secreting signal substances, the SMS cells described herein andcells derived therefrom, displace the position of cells by physicallycarrying them.

In various embodiments, the SMS cell line exhibits exceptionalcharacteristics, including but not limited to: robust growth in standardmedium for more than 30 passages, resistance to adverse conditions,growth in absence of serum, formation of cell culture multilayer,capacity for differentiation into different specialized cells, whichmakes it most ideal for autologous cell therapy. In some embodiments,the SMS cell lines also excrete heavily extra cellular matrix (ECM)providing therefore an important source of this valuable substances.

Production of ECM, makes SMS cell type a candidate supportive cell forproductive coculturing with other cells. SMS cell line demonstratesfurther an extraordinary ability for forming complex multicellularmacrostructures in vitro. These macrostructures resemble those observedin human and animal tissues and organs, making it a potential richesource for autologous tissue replacement.

In various embodiments, provided herein are isolated cell linecomprising Small Mobile Stem cells (SMS) and uses thereof.

In some embodiments, the SMS cells have a size of less that about 10 μm,or less that about 8 μm, or less that about 6 μm, or between about 3 μmto about 6 μm, or between about 4 μm to about 6 μm, or between about 4.5μm to about 6.5 μm.

The SMS cells disclosed herein maintain a geometrically definedcharacteristic for the nucleus, allowing them to be differentiated fromother cells without the need for one or more probes.

In various embodiments, the SMS cells exhibit a translucent-likecytoplasm. In some embodiments the SMS cells also exhibit a circularnucleus (2.5-3.5 μm) that includes a centrally located very small circle(1-1.5 μm) of different light contrast. In specific embodiments, thecircular nucleus is about 2.5-3.5 μm and includes a centrally locatedvery small circle with a size of 1-1.5 μm and having a different lightcontrast. In these embodiments, depending on focusing plane, the minutecircle appears darker or lighter. These contours of the cell bearresemblance to the shape of a human “Iris” and, for cells that remainundifferentiated, are stable throughout cell culture.

In some embodiments, the undifferentiated SMS cells do not stain withstandard staining techniques. Non-limiting examples of standard stainingtechniques include Wright's stain, Coomassie blue, Neutral red,Safranin, Silver staining, Masson's trichrome, Amido black, Toluidineblue, methylene blue, PAS staining, Trypan-blue, and Haematoxylin. Inother embodiments, the cell that remains undifferentiated is stablethrough cell culture.

In various embodiments, the speed of moving SMS cells reaches about 1.5μm/sec; which is about three times faster than the fastest reported cellof human origin: the neutrophils (a maximum speed of 0.5 μm/sec). See,e.g., Stossel, T. P. “The E. Donnall Thomas Lecture, 1993. The machineryof blood cell movements”, Blood 84.2 (1994):367. In some embodiments,the high mobility speed of the SMS cells is greater than about 0.5μm/sec. In specific embodiments, the high mobility speed is about 1.5μm/sec.

In some embodiments, the SMS cells are expressive of extracellularmatrices in standard media, including but not limited to DMEM lowglucose, DMEM high glucose, with or without Hepes and/or serum.

In various embodiments, the SMS sells are capable of forming anorganized extracellular matrix (ECM). In these embodiments, theformation of the organized ECM makes the cells amenable for practicalapplications, including but not limited to industrial applications aswell as medical applications. The ability of the SMS cells to produceECMs is unique to these cells and allows for the production of aprotein.

The SMS cells disclosed herein are sensitive to environmental changeswhile being resistant to adverse conditions. For example, the SMS cellsare sensitive to media compositions conducive to differentiation, aswell as changes to PH and temperature. These same cells are resistant toadverse conditions that cause death to other human cells including butnot limited to elevated or reduced temperatures, PH, ionic strength,freezing and thawing in growth medium. For example, various embodimentsof the SMS cells described herein can be frozen and then thawed withoutthe need for a protectant.

In some embodiments, the media is DMEM low glucose or DMEA high glucose,optionally with Hepes or serum. In other embodiments, the environmentalchange is a media composition conducive to differentiation, a change tothe pH, an elevated temperature or a reduced temperature and the adversecondition that causes death to other human cells is freezing and thawingin a growth medium, ionic strength, elevated or reduced temperature, orpH. In specific embodiments, the elevated temperature is about 90° C.and the reduced temperature is less than about −20° C. In other specificembodiments, the pH is between about 6 to 9.

Various embodiments of the SMS cells are robust and highly proliferativein standard media. For example, a single SMS cell line maintains a stateof continuous growth for at least one 1 year, at least 2 years, at least3 years, at least 4 years, or between 1-5 years, between 1-4 years, orbetween 2-3 years.

In various embodiments, the SMS cell lines can be grown in a basalmedium without serum or special additives, including but not limited togrowth factors. These cells are also responsive to differentiationinductive media efficient for differentiation of other types of cells.

In various embodiments, provided herein is an isolated cell linecomprising: Small Mobile Stem cells (SMS), wherein the SMS: (i) have asize of between about 4.5 to about 5.5 mm; (ii) have a regularcharacteristic nuclear shape and a largely translucent cytoplasm; (iii)have an exceptionally high mobility speed; (iv) are expressive ofextracellular matrix in a standard media; (v) form an organized ECMlayer and cellular multilayers; (vi) are sensitive to environmentalchanges and resistant to adverse conditions that cause death to otherhuman cells; (vii) are robust and proliferative in standard media formore than 30 passages; (viii) can be grown in absence of serum andspecial additives; and (ix) are responsive to common differentiationinductive media.

In some embodiments, the regular characteristic nuclear shape includes acircular nucleus. In specific embodiments, the circular nucleus is about2.5-3.5 μm and includes a centrally located circle of about 1-1.5 μm.

In various embodiments, the original cells are isolated from umbilicalcord blood, peripheral blood, bone marrow, or a solid tissue. Specificnon-limiting examples of solid tissue useful in the invention describedherein are placenta, liver, heart, brain, kidney or gastro intestinaltract.

In some embodiments, the SMS cells form a strong attachment to plasticand a poor attachment to normal glass.

In other embodiments, the SMS cells readily form multi-functionalcomplex assemblies of cells.

In some specific embodiments, the SMS cells are successfully cultured inan osteogenic differentiation medium and assemble into complexstructures.

In some specific embodiments, the SMS cells act as Peelers (i.e., peel arectangular portion of the ECM membrane). In these embodiments, the SMSderived cells initially cut the connective membrane to variable distanceand then cut at the two ends at fixed angles to the prior incision forextended lengths. In these embodiments, each of the duplicate membraneincisions continue to proceed parallel in an equidistant mode resultingin a long stretch of rectangular membrane with constant diameter.

In other specific embodiments, the SMS derived cells act as coaters(i.e., are generators of flat membranes, are surface crawlers and appearto be multi-surface feeders). In these embodiments the SMS cells aregenerators of flat membranes and appear to be multi-surface feeders.Based on the shape, they appear as surface crawlers and may haveextended and oriented sensing.

In some specific embodiments, the SMS cells and cells derived therefromact as liners (i.e., initiators of extensive linear cell assembly). Inthese embodiments, the SMS cells are initiators of multicellularextensive linear assembly.

In other embodiments, the SMS cells and cells derived therefrom act asweavers (i.e., pick Longitudinal Flat SMS derived cells (LFS) assemblycells and reposition those diametrically). These cells exhibit variousshapes and sizes.

Also provided herein are methods for treating or preventing a disease orcondition comprising introducing SMS cells described herein into asubject. In some specific embodiments, the disease or condition is aliver disease, kidney disease, cardiovascular disease, diabetes, cancer,burn, spinal cord injury, retinal disease, arthritis, genetic defect,neurologic disease or immune-mediated disease. In other specificembodiments, the cancer is leukemia or lymphoma; the neurologic diseaseis a neurodegenerative disease or condition. In various embodiments, theneurodegenerative disease or condition is Parkinson's, Amyotrophiclateral sclerosis or Alzheimer's disease.

Also provided herein are methods for treating or preventing age relatedconditions comprising introducing SMS cells described herein to asubject. In specific embodiments, the age related condition is cosmeticin nature.

In some embodiments, provided herein are methods for preventing the lossof or generating new functional cells to repair a tissue or organ in apatient by administering SMS cells described herein.

In other embodiments, provided herein are methods for testing the safetyand efficacy of a drug in a patient comprising preparing an isolatedcell line described herein, exposing the isolated cell line to a drug,and evaluating the safety or efficacy of the drug in the cell line.

Also provided herein are industrial uses of the cell lines describedherein. In some specific embodiments, the SMS cells are useful for theproduction of one or more proteins. In other specific embodiments, theSMS cells are useful for the production of a compound.

Other features and advantages of the present disclosure will become morereadily apparent to those of ordinary skill in the art after reviewingthe following detailed description and accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The details of the present invention may be gleaned in part by the studyof the accompanying drawings, in which:

FIG. 1 exemplifies, images of cultured SMS cells, derived from UCB, withtypical circular shape of the nucleus (˜3 μm) and the small dark circlein the middle of the nucleus, according to one embodiment of theinvention. In FIG. 1(a), the cytoplasm is hardly visible as a shadow.FIG. 1(b) illustrates SMS cultured multi-cellular layers appearingopaque. At the bottom of a flask; extracellular matrix made by in vitrocultured SMS cells, stained with safranin are illustrated. In FIG. 1(c),the three arrows point to the distinctive three sub-layers.

FIG. 2 exemplifies, the path of the moving SMS cell as tracked by theTracker software and according to one embodiment of the invention. Thewhite arrows in the image point to single cells and cell clusters.Illustrated in the bottom box is a plot diagram of movement timerelative to X coordinate position of the cell (circled).

FIG. 3 illustrates that the Coculture of SMS cells (small circularcells) and human red blood cells (large circular cells) both exhibit thesurface invagination (arrows) detected as contrast light variationsthrough microscopically varying the focusing plane (FIG. 3A). In vitrocell culturing demonstrating self organization of SMS cells and SMSderived cells resulting in complex assembly of various specificstructures. In these embodiments there is a strait cell alignment byanisotropic aggregation (FIG. 3B). Amido black staining of slidepreparations showing a complex multilayered structure that includesmulti-membranes associated and/or concomitant with SMS and SMS derivedcells (FIG. 3C).

FIG. 4 exemplifies, SMS cells derived from UCB cultured in osteogenicdifferentiation medium, according to some embodiments of the disclosure.Assembly into complex structures; the adherent fraction (FIG. 4A); thesupernatant fraction (FIG. 4B). Structures, stained with alizarin Red S(FIG. 4C). FIGS. 4D, 4E and 4F illustrate various complex structuresderived from the supernatant fraction.

FIG. 5 exemplifies, SMS cells derived from UCB; cultured in adipogenicdifferentiation medium according to some embodiments. The shape of cellsindicates drastic morphological changes (FIG. 5A), assembly of cellsinto complex structures (FIG. 5B). Complex tissue-like structuresstained with Oil Red O are illustrated in FIG. 5C.

FIG. 6 illustrates SMS cells derived from UCB in cultured neurogenicdifferentiation medium (FIG. 6A, FIG. 6B) and cell assembly followingearly induction (FIG. 6C). The shape of various silver stained cellsappearing black or dark brown is consistent with neuronal silverstaining (FIG. 6D, FIG. 6E). Assembly of differentiated cells afterseveral weeks of induction is illustrated in FIG. 6F.

FIG. 7 exemplifies parallel duplicate membrane incisions is initiated bya diametrical incision that may vary in length (1) causing the membraneto expand diametrically; and variation in length through parallelconcomitant incision (2, 3), according to some embodiments of thedisclosure.

FIG. 8 exemplifies, Parallel duplicate membrane incisions: SMS derivedcells cut the connective membrane to variable distances and proceed induplicate parallel membrane incisions continue to proceed parallel in anequidistant mode (arrow) (FIG. 8A, FIG. 8B), resulting in a long stretchof rectangular membrane with constant diameter. In this embodiment,several of the duplicate incisions proceeding at various distances, areactually extending parallel to each other (arrows) (FIG. 8A). Differentfolding pattern and mostly spiral folding is observed for the partiallycut membrane (arrow heads) (FIGS. 8A, 8B, 8C, and 8E). Cells coating theliberated membrane (arrows) convert this structure into what appears tobe a tubular structure (FIG. 8D, FIG. 8E). In some cases the membrane iseven embedded within membrane during this process (arrow) (FIG. 8F). Allimages were taken from living cultured cells.

FIG. 9 exemplifies, three dimensional cluster mesh assembly: Amido blackstained slide preparations showing aggregation of SMS cells that form acore with disorderly branches (FIG. 9B, FIG. 9C). The circular discoidshaped cells appear to aggregate side by side (FIG. 9C). SMS cellsreshape converging their circular invaginations (see arrows at FIG. 9C)forming a continuous groove in associated cells (FIG. 9D, FIG. 9E).Folding (wrapping) of associated cells forms fine tubes of ˜3-4 μmdiameter. Branches connect and fuse (anastomose) forming a mesh (FIG.9F, FIG. 9G). For comparison, images of aggregated living cells in acell culture flask are provided (FIG. 9H, FIG. 9I).

FIG. 10 illustrates a coated tubular assembly according to someembodiments: SMS cells reshape enlarging and forming round cells ofdarker tone (arrow 1), these cells flatten and extend to becomelongitudinal (EFS cells) (FIG. 10A); two EFS cells associate (arrow 2)(a); partial and complete wrapping of EFS cells (FIG. 10B, FIG. 10D);extension in length by cellular associations (arrow) (FIG. 10C); coatingEFS cells by SMS derived cells (FIG. 10E, FIG. 10F); mobilization of EFScells into a growing coated tube (FIG. 10G); the growing tube showsbranching (FIG. 10H). In some embodiments, large extended tubes separatefrom the adherent fraction of the flask and appear attached to eachother through membrane (FIG. 10I). All images were taken from live cellsin culture.

FIG. 11 exemplifies EFS cell assembly able to undergo fusion that mayoccur: juxtaposed side by side (1) resulting in a diametrically largertube; tip to tip (2) resulting in an elongated tube; tip to side (3)resulting in branching, according to some embodiments of the disclosure.

FIG. 12 exemplifies the two dimensional leaf mesh assembly involvesextension of a thick core structure and perpendicular primary andsecondary branching, according to some embodiments of the disclosure.

FIG. 13 exemplifies a two dimensional leaf mesh assembly according tosome embodiments: amido black staining of SMS cell aggregate forming acore with perpendicular branches (FIG. 13A, FIG. 13C, FIG. 13D); primarybranches form thinner perpendicular secondary branches (FIG. 13E); theassembly mature (anastomose) into an interconnected mesh; visualized inslide preparations using both amido black staining (FIG. 13F) andtoluidine blue (FIG. 13G); Masson Goldner trichrome staining ofresulting leaf mesh assembly (FIG. 13H, FIG. 13I). In some embodiments,the same structure is partially visualized using Masson Goldnertrichrome staining in culture flask, indicating the presence of hiddenleaf mesh assembly (FIG. 13B).

FIG. 14 exemplifies, crescent formation, membrane tube transition andfenestrations according to some embodiments: Crescent formations appearat the edges of the leaf mesh assembly (arrow) (FIG. 14A); these containEFS cells that migrate away from the assembly while digesting a membranelayer (FIG. 14B); crescent formations migrate into assemblies and fusinginto larger crescents as shown by amido black staining (FIG. 14E, FIG.14F) and toluidine blue staining (FIG. 14G, FIG. 14H). In someembodiments, these represent pretubular membrane folding which are cutout (Figured 14I, 14J, 14K, 14L). Some crescent formations combine toform fenestrations within the membrane (window like structures) thediameters of which varies (FIG. 14I, FIG. 14J).

FIG. 15 illustrates images of slide preparations of stained sheep hearttissues according to some embodiments. The structures are consistentwith the observation in three dimensional cluster mesh assembly (FIG.15A) in Coated tubular assembly (FIG. 15B) in two dimensional leaf meshassembly (FIG. 15C, FIG. 15D) in Crescent formation (FIG. 15E) inmembrane tube transition (FIG. 15F, FIG. 15G) and fenestrations (FIG.15H, FIG. 15I). The images (FIGS. 15A, 15C, 15E, 15F, 15I) were takenfrom slide preparations stained using amido black stain, images (FIGS.15B, 15D, 15G, 15H) were from slide preparations stained using theMasson Goldner trichrome technique.

FIG. 16 illustrates a white layer at the bottom (FIG. 16E) which isactually crammed with SMS cells. The image FIG. 16E is digitallyenhanced and subjected to inversion (FIG. 16F).

FIG. 17 illustrates amido black stained rat Cerebellum. The black dotsindicate the presence of SMS cell like shaped cells in abundance.

FIG. 18 illustrates Masson Goldner trichrome stained rat Cerebellum SMScell like shaped cells appear abundant on the white background (arrowwith dashes). Further cells of similar shape but that are larger and arestained appear derived from SMS cells (straight arrow).

FIG. 19 illustrates amido black stained rat heart indicate the crammedpresence of SMS cell like shaped cells. Cells are positionally graduallyreplaced by larger differentiated and stained cells (arrow). Image wassubjected to digital inversion.

FIG. 20 illustrates amido black stained rat heart indicate the presenceof SMS cell-like shaped cells. Cells that are indicated by the blackdots (FIG. 20A) are made more visible by subjecting image to digitalinversion (FIG. 20B).

FIG. 21 illustrates amido black stained rat heart indicate the crammedpresence of SMS cell like shaped cells. Image was subjected to digitalinversion.

FIG. 22 illustrates Masson Goldner trichrome stained rat heart indicatethe crammed presence of SMS cell like shaped cells. Some cells vary onlyslightly from the SMS cell shape geometry but demonstrate intenselydifferent pigmentations or different affinities to stain.

FIG. 23 illustrates Masson Goldner trichrome stained rat heart indicatethe clear presence of cells with the characteristic SMS cell like shape.In this embodiment, some cells appear to be dividing.

FIG. 24 illustrates Masson Goldner trichrome stained rat heart indicatethe crammed presence of SMS cell like shaped cells. In this embodiment,some cells vary only slightly from the SMS cell shape geometry butdemonstrate intensely different pigmentations or different affinities tostain.

FIG. 25 illustrates Masson Goldner trichrome stained rat kidney indicatethe presence of SMS cell like shaped cells.

FIG. 26 illustrates amido black stained rat liver indicate the presenceof SMS cell like shaped cells. Cells are positionally gradually replacedby larger differentiated and stained cells (arrow).

FIG. 27 illustrates amido black stained rat liver indicate the crammedpresence of SMS cell like shaped cells.

FIG. 28 illustrates Masson Goldner stained rat liver indicate thecrammed presence of SMS cell like shaped cells.

FIG. 29 illustrates Masson Goldner stained rat liver indicate thecrammed presence of SMS cell like shaped cells.

FIG. 30 illustrates Masson Goldner stained rat liver tissue indicate thecrammed presence of SMS cell like shaped cells. Image was subjected todigital inversion.

FIG. 31 illustrates toluidine blue stained sheep cerebellum tissueindicate the crammed presence of SMS cell like shaped cells. Cells arepositionally gradually replaced by larger differentiated and stainedcells (arrow) (FIG. 31A). Image of FIG. 31A was subjected to digitalinversion for better distinction of SMS cell like shaped cells (FIG.31B).

FIG. 32 illustrates toluidine blue stained sheep cerebellum tissueindicate the presence of SMS cell like shaped cells.

FIG. 33 illustrates amido black stained sheep heart tissue indicate thecrammed presence of SMS cell like shaped cells.

FIG. 34 illustrates Masson Goldner trichrome stained sheep heart tissueindicate the presence of SMS cell like shaped cells. Aggregated cellsproducing extracellular matrix appearing identical the one produced bySMS cells in vitro (arrows) (see, FIG. 13H, FIG. 13I from SMS cellculture).

FIG. 35 illustrates Masson Goldner trichrome stained sheep liverindicate the crammed presence of SMS cell like shaped cells.

FIG. 36 illustrates Masson Goldner trichrome stained sheep liverindicate the crammed presence of SMS cell like shaped cells.

FIG. 37 illustrates Masson Goldner trichrome stained sheep liverindicate the crammed presence of SMS cell like shaped cells.

FIG. 38 illustrates Toluidine blue stained sheep liver indicate thecrammed presence of SMS cell like shaped cells.

FIG. 39 illustrates Masson Goldner trichrome stained sheep kidneyindicate the crammed presence of SMS cell like shaped cells. Cells arepositionally gradually replaced by larger differentiated and stainedcells (arrow).

FIG. 40 illustrates the detection of the neuron specific antigen betatubulin using antigen specific antibody in SMS cells subjected to invitro neuronal differentiation.

FIG. 41 illustrates the detection of the neuronal cells specific antigenGFAP using antigen specific antibody in SMS cells subjected to in vitroneuronal differentiation. Image (a) was subjected to enhancement anddigital inversion.

FIG. 42 illustrates the detection of the neuronal cells specific antigenPLP using antigen specific antibody in SMS cells subjected to in vitroneuronal differentiation. FIG. 42A was subjected to enhancement anddigital inversion.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

After reading this description it will become apparent to one skilled inthe art how to implement the invention in various alternativeembodiments and alternative applications. However, although variousembodiments of the present invention will be described herein, it isunderstood that these embodiments are presented by way of example only,and not limitation.

The invention described and elaborated herein refers to by us discoveredhuman Small Mobile stem cell line (SMS) (Rahmo et al (2013) 1 Journal oflife sciences and technologies 56-61) its isolation, and itsdifferentiation into other cell types and to multicellularmacrostructures. While the present application is exemplified using theSMS stem cell line derived from the human cord blood and from peripheralblood, this is not intended to limit the scope and subject of thepresent application and those skilled in the art will recognize that thenovel stem cell type its isolation, differentiation and resultingcomplex cellular macrostructures described herein may be derived from avariety of different tissue sources that include SMS cells.

The present invention arose from the present inventors initialobservations of very small sized adherent cells (˜5 μm) in a standardcell culture system. These uniquely shaped SMS cells wereequi-dimensional, with strict radial symmetry. They exhibit in a lightmicroscope a translucent cytoplasm, and a circular nucleus (˜3 μm) thatincludes a centrally located very small circle (˜1 μm) of differentlight contrast. Depending on focusing plane, the minute circle appeareddarker or lighter. These, throughout cell culture, stable contours ofthe small cell bear resemblance to the shape of a human “Iris” (see,e.g., FIG. 1A). The observed concentric inner circle appears to be aresult of cell surface invagination. This observation is comparable towhat is microscopically perceived for the biconcave surface structure ofred blood cells (see, e.g., FIG. 3A).

SMS cells were clearly distinguished following 3-4 weeks of cell cultureinitiation. Cells with the described appearance did not stain with anyof the standard Staining techniques (giemsa, methylene blue, Papanicola,and Wright staining) (see, e.g., FIG. 3C). SMS cells strongly adhered toplastic, and detachment using trypsin recipes was unsuccessful. Cellswere therefore dislodged using a scraper or through cold shocktreatment. Primary culture cells reached confluency in 3-4 weeks. SMScells were able to proliferate into a multilayer. SMS cells werecontinuously cultured for over two years (30+ passages), with noapparent effect on proliferation potential.

Exploring further characteristics of SMS cell type demonstrated itsresponsiveness to common differentiation inductive media, including butnot limited to osteogenic, adipogenic and neurogenic media. Cells werecryopreserved at different passages and successfully revived in the samegrowth medium (Rahmo et al (2013) 1 (1) Journal of life sciences andtechnologies 56-61).

The present invention was not anticipated based upon the current stateof the art. SMS cell type has a unique overall shape (size and theparticular form) among reported human stem cells and other reportedhuman cells.

Cell Separation

SMS cells from whole blood were isolated based mainly on strong plasticadherence. Mononuclear cell fraction was recovered by diluting each 10mL UCB sample with an equal volume of PBS, layering over an equal volumeof Ficoll-Paque (1.077 g/ml) (GE health care Biosciences AB, Sweden).Mononuclear cells (MNCs) from the gradient interface were washed twicewith PBS after centrifugation at 400 g for 30 min. The layer ofsedimented red blood cells was also collected; it represented the RBCfraction. The cell fraction positive for CD34 was isolated from the MNCfraction based on magnetic microbead selection procedure. High-gradientmagnetic field and MiniMACS (MS) columns were used (Miltenyi Biotech,Germany).

Cell Isolation and Culture

Whole UCB was used for culture after testing negative for bacterial orfungal contamination. The adherent heterogeneous population of cells wasobserved on days: 4 and 5. Through continuous change of medium, thesuspended cells became gradually fewer. Cells of various shapes appearedat the bottom of culture flasks. The SMS cells were observed as wasdescribed in Rahmo A., et al.: Introducing a novel human stem cell withexceptional characteristics: Small, Mobile Stem Cells (SMS). JOLST 2013,1: 56-61.

Primary culture cells reached confluence in 3-4 weeks. SMS cells wereable to proliferate into a multilayer. SMS cells were continuouslycultured, starting 11-2010 till 7-2012 and further (27+ passages), withno apparent effect on proliferation potential. Cell separation usingFicoll density gradient centrifugation, indicated that SMS cells areenriched in the RBC layer. The same cells however have been alsoisolated from the MNC layer; mainly as a CD34+ fraction.

Testing the presence of SMS cells in blood of animals, including but notlimited to rats, using essentially the same methods of isolationresulted in the same outcome observed for human tissue. Further evidenceof the presence of SMS cells we obtained from the dissection of varioustissues from various organs of Rat and Sheep. See, e.g., FIGS. 17-30(rat) and FIGS. 31-39 (sheep).

Resistance to Adverse Conditions

SMS cell line demonstrated extraordinary resistance to variousnon-physiological conditions. These include low and high temperature,freezing and thawing at −20° C. in standard growth medium (Low-glucoseDulbecco's Modified Eagle's medium LG DMEM (Gibco) with GlutaMAX™ andsupplemented with 10% heat inactivated fetal bovine serum (FBS)(Invitrogen), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco)),dehydration, high PH values, and variations in ionic strength.

SMS cell line were grown in absence of any added serum that is in(Low-glucose Dulbecco's Modified Eagle's medium LG DMEM (Gibco) withGlutaMAX™, 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco))incubated at 37° C. and 5% CO₂ in a humidified atmosphere using theincubator (SHEL LAB, USA) for months with no apparent effect on shape oradverse effects on proliferation potential of SMS cells.

Cell Mobility

The characteristics of cell movements are some of the parameters thatdefine specific cells. Cells vary, for instance, considerably in theirspeed; fibroblasts move very slowly (12-60 μm/h), while neutrophiles,thought to be the fastest moving human cell, reach speeds of 30 μm/min(Persson et al. (2010) “Cell motility studies using digital holographicmicroscopy,”. in: Microscopy: Science, Technology, Applications andEducation. A. Méndez-Vilas, J. Diaz, Ed. FORMATEX 1063, Ch 35.).

Cell movement is of particular importance to issues of organogenesis,tissue repair and malignant cell invasion. Fast cellular movements weremicroscopically observed real time in some cells; that is during normalcell culture conditions. Cell speeds of about 1.5 μm/sec were readilymeasured (see, e.g., FIG. 2). Such fast cellular movements can beinduced in almost the entire cell population; that is by applying whatcan be categorized as adverse cellular conditions (PH shock, cold chock,dislodging). Cellular movement was also observed in cell culturescontaining osteogenic inductive media; in this case whole clusters ofcells appeared to be dragged by single cells. The motile features of SMScell line were quite surprising, since obtained speed values exceededthat of neutrophiles; they are therefore an important defining featureof SMS cells.

Cell movements include also movements characterized by a cell clustermoving collectively (i.e., remaining in contact during movement).Collective movement is now widely recognized in embryogenesis and cancer(Mayor et al. (2010) 20 (6) Trends Cell Biol 319-28). SMS fast movingcells allow observation in short time intervals; in which holdingculture parameters constant is easily achieved. Accordingly, they serveas an important model, amenable for studying such complex tissue patternforming movements (Rahmo et al (2013) 1 (1) Journal of life sciences andtechnologies 56-61).

Multilayer Formation

SMS cell lines display a robust proliferative potential with formationof cellular multilayer in vitro using the growth medium including serumand the one excluding serum and in the absence of any added substancesuch as matrigel or cell extracellular matrix proteins.

Extensive ECM Production

SMS cell lines are able to co-synthesize organized layers of ECM duringextensive cell proliferation. Following few weeks of cell culturing ingrowth medium, a complex organization of cells and extracellular matrix(ECM) was observed. Some of these structures are organized as layers ofmembranes that include SMS cells, SMS derived cells, and complex cellassemblies (see, e.g., FIG. 1C, FIG. 3C, FIG. 16E, FIG. 16F).

Three dimensional polymers mimicking ECM, as well as reconstitutedbasement membrane, such as commercial product Matrigel, have been shownto promote cell organization and differentiation (Gwendolen et al.(2010) 43 (1) Journal of Biomechanics 55-62).

Matrix topography and its physical characteristics are proven to beinfluential parameters. It is thought that at 3D culture conditioncellular gene expression would mirror more closely the in vivo state.Applications included the monitoring of tumor growth and metastasis,tissue engineering, and organ printing.

Macrostructures

Regular multicellular pattern formation was observed during cellculture; especially in the presence of inductive media. Non-limitingexamples of inductive media useful in various embodiments of the presentinvention include: a Neurogenic Differentiation Medium such as: 1 mMb-mercaptoethanol (BME) added to growth medium for 24 h followed bytreatement with 2% DMSO and 200 mM butyrate hydroxyanisole (BHA) ingrowth medium without Fetal bovine serum (see, e.g., X. Q. Kang, W. J.Zang, L. J. Bao, D. L. Li, X. L. Xu, and X. J. Yu, Differentiatingcharacterization of human umbilical cord blood-derived mesenchymal stemcells in vitro, 1 Cell Biology International, vol. 30, pp. 569-575, July2006); an Adipogenic Differentiation Medium such as: growth medium plus:1 μmol/Ldexamethasone, 5 μg/mL insulin, 0.5 mM isobutylmethylxanthine,and 60 μM indomethacin (see, e.g., A. Erices, P. Conget, and J. J.Minguell, Mesenchymal progenitor cells in human umbilical cord blood,British Journal of Haematology vol. 109, no. 1, pp. 235-242, April2000); and/or an Osteogenic Differentiation Medium such as: growthmedium plus: 0.1 μM dexamethasone, 0.05 mM ascorbic acid-2-phosphate and10 mM β-glycerophosphate (see, e.g., N. Jaiswal, S. E. Haynesworth, A.I. Caplan, and S. P. Bruder, Osteogenic differentiation of purified,culture-expanded human mesenchymal stem cells in vitro, J Cell Biochem.vol. 64, no. 2, pp. 295-312, February 1997).

Such complex patterning at individual and multicellular level suggestsmultiple or extensive changes at gene expression status; echoing theobserved gross changes to individual cell architecture and multicellularorganization.

The applied standard inductive media resulted in a multitude of clearlydistinctive complex multicellular phenotypes. These observations outlinethe organo-genetic potency of SMS cells. The development of thesestructures is a continuous dynamic process that requires weeks andinvolves typical tissue forming cell activities: cell division(symmetric and asymmetric), cell aggregation, and cell differentiation.

Moreover, cells appear to migrate in a coordinated fashion, tocooperate, and to associate spontaneously, forming complex cell-ECMstructures of various compositions; some of which are transient (see,e.g., FIG. 3B).

Fractions of tissue like structures and its components may detachspontaneously from the adherent fraction of the flask and were examinedin the supernatant suspension of the cell culture. The main fractionremained adherent to the flask, and was detached by scraping forsubculturing or examination (Rahmo et al. (2013) 1 (1) Journal of lifesciences and technologies 56-61).

Cryopreservation

A Trypan-blue dye exclusion test was used to distinguish the cellviability (Invitrogen). Cell number and viability were estimated usingthe Countess® cell counter (Invitrogen). Cells were cryopreserved usinga freezing medium consisting of 60% growth medium with 30% FBS and 10%DMSO.

The present invention teaches the presence of a new cell type withexceptional characteristics. This cell type is very small in size, has aregular characteristic nuclear shape and a very translucent cytoplasm.SMS cell lines are very fast in terms of mobility, but attach verystrongly to plastic and very poorly to normal glass. They do not stainwith many standard staining techniques. SMS cell lines proliferate instandard media for more than 30 passages, are quite resistant to adverseconditions, form a multilayer, excrete ECM, can be grown in absence ofserum and have a capability of forming complex multicellularmacrostructures. The exceptional characteristics of this cell typeassert clearly its novelty among earlier described stem cells.

Indeed, this novel cell line provide significant advances of the priorstate of the art. Although many different stem cell lines and processesfor their isolation and utilization exist and are being examined, theprocedures related to them have shortcomings. For example, none of themprovide the following SMS cell line advantages: (1) the ability toisolate SMS cells from various organs including cord blood andperipheral blood; (2) the ability to proliferate for extensive longperiod of time in modest media; (3) the ability to proliferate in mediasupplemented with human serum or without any added serum (basic mediumwithout additives); (4) resistance to adverse conditions (such as lowand high temperature, pH changes, dehydration); (5) the ability toprovide for autologous transfer of these stem cells, and cells derivedfrom it, in high quantity; (6) the ability to differentiate intomulti-cellular complex structures; (7) the ability to provide forautologous transfer of differentiated tissue, identical or tissue likestructures produced in vitro; and (8) the ability to synthesize andproduce extracellular components of potential therapeutic and industrialuses.

EXAMPLES Example 1 Cell Culture and Cryopreservation

Cells from whole peripheral blood, whole umbilical cord bloodmononuclear fraction and RBC fraction were cultured separately andallowed to adhere to the bottom of the T25 flask (Techno Plasticproducts TPP).

Culture was initially incubated in growth medium for four days. Mediumchanges were carried out twice weekly thereafter. Growth medium used wasLow-glucose Dulbecco's Modified Eagle's medium LG DMEM (Gibco) withGlutaMAX™ and supplemented with 10% heat inactivated fetal bovine serum(FBS) (Invitrogen), 100 U/mL penicillin, and 100 μg/mL streptomycin(Gibco) incubated at 37° C. and 5% CO₂ in a humidified atmosphere usingthe incubator (SHEL LAB, USA).

At later stages only half the medium was weekly changed. Cells wereusually passaged upon reaching 80% to 90% confluency using a scraper orcold shock treatment (˜1 h at 4-8° C.). Primary culture cells reachedconfluency in 3-4 weeks. SMS cells were able to proliferate into amultilayer (at least three layers) (see, e.g., FIG. 1C).

Lower layer cells became attached more strongly to the surface of theflask through a layer of extra cellular matrix (ECM). Cells werecryopreseved at different passages in cryopreserving medium (60% growthmedium, 30% added FBS, 10% DMSO) and successfully revived in the samegrowth medium.

Example 2 Osteogenic Differentiation

Following treatment with inductive medium, cells grown from umbilicalcord blood, were subcultured in growth medium (Low-glucose Dulbecco'sModified Eagle's medium LG DMEM (Gibco) with GlutaMAX™ and supplementedwith 10% heat inactivated fetal bovine serum (FBS) (Invitrogen), 100U/mL penicillin, and 100 μg/mL streptomycin (Gibco) incubated at 37° C.and 5% CO₂ in a humidified atmosphere using the incubator (SHEL LAB,USA)) for 24 hours and then subjected to osteogenic medium (growthmedium plus: 0.1 μmol/L dexamethasone (Sigma), 0.05 mmol/L ascorbicacid-2-phosphate (Sigma) and 10 mmol/L β-glycerophosphate (Sigma))(Jaiswal et al. (1997). 64 (2) J Cell Biochem 295-312).

Medium changes were performed every 3 to 4 days. The media, containingdetached cells and tissues of each flask, were centrifuged. Collectedpellets were separately cultured at the same conditions, using a tissueculture flat tube (TPP); representing the supernatant fraction. Afraction of cells changed drastically their shape and size; becominglarger and exhibiting standard cell morphology, with clear nuclei andcytoplasma.

A percentage of cells (˜30%) aggregated and tended to lessen attachmentto surface. Some aggregated cells formed assemblies of different shapes,with or without color (see, e.g., FIG. 4a ). A fraction of aggregatedcells gained a clear orange color and formed a complex assembly thatbecame brownish (see, e.g., FIG. 4A).

Many of these, and some rather complex structures, were present in thesupernatant fraction (see, e.g., FIG. 4B). This differentiation mediuminduced apparently the formation of several different cell assemblies(see, e.g., FIG. 4D, FIG. 4E, FIG. 4F). Many cells were stained stronglywith the dye Alizarin Red S.

Some of the complex assemblies of cells were also stained; indicatingaccumulated calcium deposits (see, e.g., FIG. 4C). The staining patternwas comparable to the one observed in a sheep bone control sample.Throughout culture, a large fraction of cells maintained the preinductive shape of SMS cells and continued to proliferate. Following sixweeks of incubation with inductive medium, cells were reincubated withthe original growth medium. This caused disassembly of aggregated cells,and disappearance of some earlier formed complex structures (within ˜2weeks).

Example 3 Adipogenic Differentiation

Inductive medium, cells grown from umbilical cord blood, weresubcultured in growth medium at standard conditions (Low-glucoseDulbecco's Modified Eagle's medium LG DMEM (Gibco) with GlutaMAX™ andsupplemented with 10% heat inactivated fetal bovine serum (FBS)(Invitrogen), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco)incubated at 37° C. and 5% CO₂ in a humidified atmosphere using theincubator (SHEL LAB, USA)) for 24 hours, and then grown in adipogenesismedium (growth medium plus: 1 μmol/L dexamethasone, 5 μg/mL insulin, 0.5mmol/L isobutylmethylxanthine, and 60 μmol/L indomethacin) (Erices et al(2000) 109 (1) British Journal of Haematology 235-242).

Half the medium was changed twice weekly. The media of each flask werecentrifuged. Collected pellets were separately cultured at the sameconditions using a tissue culture flat tube (TPP); representing thesupernatant fraction. Inductive medium caused many SMS cells to becomelarger; starting day 2, and some aggregated (see, e.g., FIG. 5A).

Some cells appeared to accumulate lipid droplets of yellow color. OilRed O staining, demonstrated a rich lipid deposit in some cells.Aggregated cells formed multicellular assemblies with fat deposits (see,e.g., FIG. 5b ). Many of these were present in the supernatant fraction(see, e.g., FIG. 5c ). After 10 days, flask inductive medium wasreplaced with the original growth medium causing the former phenotype todisappear (2 weeks). Cells appeared to have reversed back to theoriginal pre-inductive morphology.

Example 4 Neurogenic Differentiation

Cells grown from (peripheral and umbilical cord blood) were seededseparately in T25 flasks (TPP). At 70% confluency, 1 mMbeta-mercaptoethanol (BME) was added to growth medium at standardsconditions (Low-glucose Dulbecco's Modified Eagle's medium LG DMEM(Gibco) with GlutaMAX™ and supplemented with 10% heat inactivated fetalbovine serum (FBS) (Invitrogen), 100 U/mL penicillin, and 100 μg/mLstreptomycin (Gibco) incubated at 37° C. and 5% CO₂ in a humidifiedatmosphere using the incubator (SHEL LAB, USA)) for 24 h. Cells werewashed with D-Hanks buffer solution three times, and treated with 2%DMSO and 200 mMbutylatedhydroxyanisole (BHA) in the same growth mediumbut without FBS (Kang et al (2006) 30 Cell Biology International569-575).

Cell morphology was observed using an eclipse TS100 (NIKON) invertedmicroscope and photographed with a cybershot (Sony) camera, in roomtemperature. Cells were cultured further in the same flask for severalweeks, using the same inductive medium. Half the medium was replacedtwice weekly.

Following treatment with β-mercapto ethanol no visible changes wereobserved. After treatment with the second inductive medium, responsivecells (˜60%) changed shape, became in some cases extended, and afraction of these cells became later thicker and refractile (see, e.g.,FIG. 6A). Some cells became larger, but maintained an approximatespherical shape. Both cells formed branches. Some appeared multi polar,(see, e.g., FIG. 6A).

Some cells have their branches reaching to other cells and appeared tocreate intercellular contact (see, e.g., FIG. 6B). Some of the drasticchanges observed were within hours; while most appeared the next day.Cells remained adherent to flask. Further incubation with inductivemedium at 37° C. and 5% CO₂ for 30 days induced more significant changesto cell shape and organization. Continuous cell growth and the formationof multi-layers were observed, despite the absence of FBS in theneuronal inductive medium.

Detaching induced cells and centrifugation resulted in a larger, clearlygray colored pellet, which was in contrast to the usual small whitepellet of SMS cells; in absence of inductive media Staining these cellsindicated the presence of various argyrophilic cells, with a shapecharacteristic to neuronal cells (see, e.g., FIG. 6D, FIG. 6E, FIG. 6F).

Some cells maintained SMS pre-inductive shape and continued toproliferate in the presence of inductive medium. At the bottom of themultilayer grown cells, a complex, dense, mainly cell free layerappeared which constituted the ECM attached to the base of the flask.Reversing post inductive cell shape by re-incubating in growth mediumwas not observed in this case

Example 5 Macrostructuring

Following few weeks of cell culturing in standard growth medium andstandard conditions (Low-glucose Dulbecco's Modified Eagle's medium LGDMEM (Gibco) with GlutaMAX™ and supplemented with 10% heat inactivatedfetal bovine serum (FBS) (Invitrogen), 100 U/mL penicillin, and 100μg/mL streptomycin (Gibco) incubated at 37° C. and 5% CO₂ in ahumidified atmosphere using the incubator (SHEL LAB, USA)) a complexorganization of cells and extracellular matrix (ECM) was observed. Someof these structures are organized as layers of membranes that includeSMS cells, SMS derived cells, and complex cell assemblies (see, e.g.,FIG. 3C).

Fractions of this tissue like structure and its components detachspontaneously from the flask and were examined in the supernatantsuspension. The main fraction remained however adherent to the flask,and were detached by scraping for subculturing or examination.

The development of these structures is a continuous dynamic process thatrequires weeks and involves typical tissue forming cell activities: celldivision (symmetric and asymmetric), cell aggregation, and celldifferentiation. Moreover, cells appear to migrate in a coordinatedfashion, to cooperate, and to associate spontaneously, forming complexcell-ECM structures of various compositions; some of which are transient(see, e.g., FIG. 3B).

Despite the complexity and diversity of the observed cell-ECMstructures, the process leading to these structures is highlyreproducible. Partial or even extensive scrapping of the flask, leads toregeneration of the same structures. Repeated scrapping of emergingcultured Cell-ECM complex assemblies and its analysis using variousstaining techniques, revealed copious details of these diverse highlycomplex and self-organized structures.

Samples were fixed with 4% paraformaldehyde in PBS for 60 min at roomtemperature, and wetted with distilled water before staining. Stainingsolutions were prepared as follows: Staining using Amido black 10B(Merck, CI: 20470): A 1% dye solution was prepared using 7% acetic aciddistilled water solution. Staining using Toluidine blue (Reactifs RAL,CI: 52040): 0.1% in distilled water Staining was for both for 10 minutesfollowed by rinsing using either a 7% acetic acid distilled watersolution for amido black, or distilled water for Toluidine bluestaining. Masson Goldner trichrome staining solutions were purchasedfrom Carl Roth GmbH+KG co. and staining was according to instructions.

Highly reproducible structures pertaining (but not exclusively) totubulogenesis were discerned during various SMS cell derived processes.Pertinent processes are designated as: 1—parallel duplicate membraneincisions, 2—three dimensional cluster mesh assembly, 3—coated tubularassembly, 4—two dimensional leaf mesh assembly and derivatives.

Parallel duplicate membrane incisions: SMS derived cells initially cutthe connective membrane to variable distances and then cut at the twoends at fixed angles to the prior incision for extended lengths. Each ofthese duplicate membrane incisions continue to proceed parallel in anequidistant mode (see, e.g., FIG. 7), resulting in a long stretch ofrectangular membrane with constant diameter (see, e.g., FIG. 8A and FIG.8B). Noticeable is that several of these duplicate incisions thatproceeded at various distances, are actually extending parallel to eachother (see, e.g., FIG. 8A). This suggests a potential common directive.

Different folding pattern and mostly spiral folding was observed for thepartially cut membrane (see, e.g., FIG. 8A and FIG. 8E). This suggestsinternal physical forces on the membrane. However cells coating theliberated membrane convert this structure into what appears to be atubular structure (see, e.g., FIG. 8D and FIG. 8E). In some cases themembrane is even embedded within membrane during this process (see,e.g., FIG. 8F). The size of the tube may vary based on the describedprocess. Diametrical incision length, and the distance of the parallelincisions, varies tube's diameter and length.

Three dimensional cluster mesh assembly: SMS Cells enlarge slightly,most notably the small inner circle, and aggregate in an organizedcluster assembly that lay in 3D embedded within the ECM (see, e.g., FIG.9A and FIG. 9B). The core of each cluster is approximately linear,composed of single cells. From that main aggregate core, branches extendat irregular intervals and no secondary branches appeared (see, e.g.,FIG. 9C).

Cells associate topically side by side in a thread like manner (see,e.g., FIG. 9D). Cell's individual surface invaginations converge to forma continuous valley. Folding of outer edges (wrapping) would convert thethread into a potential tube (see, e.g., FIG. 9E, FIG. 9F). Threadassemblies connect (anastomose) creating a dense mesh of a regulardiameter as determined by SMS cell dimensions (see, e.g., FIG. 9F). TheSMS cells do not undergo major shape changes within this process.Various stages of this process were observed on cultured living cells(see, e.g., FIG. 9H and FIG. 9I).

Coated tubular assembly: SMS cells of radial symmetry grossly modifytheir shape, becoming larger and brownish dark. Such cells were enrichedin the CD34 positive fraction isolated by magnetic beads. The cellflattens and converts, by apparently combining with other cells ordividing, into a flat longitudinal SMS (FLS) derived cell assembly.Radial symmetry is converted hence into bilateral symmetry (see, e.g.,FIG. 10A, FIG. 10B and FIG. 10C).

Flat Longitudinal SMS derived cell assemblies (FLS) appear to apply awrapping mechanism for creating a tubular structure (see, e.g., FIG.10D). Edges of FLS cell assembly fold and connect forming a hollow core(see, e.g., FIG. 10D). SMS derived cells of differing shape areattracted to the assembly, and coat the extended tubular structure (see,e.g., FIG. 10E and FIG. 10F). The coated tubular assembly structureappears to extend by mobilizing other FLS derived cells (see, e.g., FIG.10G). Depending on FLS association pattern (sideway or at the tip),tubes may grow in length or diameter, or even extend branches (see,e.g., FIG. 10H and FIG. 11). These large structures detach readily fromthe adherent fraction and are observed floating in media. Occasionallyseveral of coated tubular assemblies are floating, attached byconnective membrane (see, e.g., FIG. 10I). This process can be easilyobserved on living cells in culture.

Two dimensional leaf mesh assembly and derivatives (membrane—tubetransition by crescent formation, fusion, and slicing, two dimensionalmembrane fenestration): SMS cells aggregate in a characteristicorganized ramification, embedded as a 2D planar complex within ECM. Thecore is mostly linear. From that core perpendicular branches at regularintervals are extending and yet smaller ones grow at regular intervalsperpendicular to primary branches (see, e.g., FIG. 12, FIG. 13A, FIG.13C, FIG. 13D, FIG. 13E). This leaf vein like formation is quiteinvisible in unstained culture flask. Merely the portions of the fixedculture flask stained with Masson Goldner trichrome method, reveal theactual presence of the leaf vein like organized assemblies (see, e.g.,FIG. 13B). The organized ramification matures into a fine mesh thatcovers a large surface area and appears rather like a network of fineconnecting tubular web (see, e.g., FIG. 13F, FIG. 13G).

Membrane—tube transition by crescent formation, fusion and slicing: Sidebranches tend to form characteristic crescent structures. They includeflat longitudinal SMS cell derived assemblies (FLS) that appear to movein targeted directions. Concomitantly, a membrane layer is beingdigested in the direction of FLS movement (see, e.g., FIG. 14A, FIG.14B, FIG. 14C, FIG. 14D). The merger of the crescent structuresestablishes large membrane folds, representing pre-tubular structuresembedded in a planar connective membrane (see, e.g., FIG. 14E, FIG. 14F,FIG. 14G, FIG. 14H). Large pre-tubular structures are further processedby slicing it out from the embedding connective membrane (see, e.g.,FIG. 14I, FIG. 14J, FIG. 14K, FIG. 14L). The result is a rather longfree nascent tube.

Chronically coordinated asymmetrical cuts, appear to be involved, andmay ensure correct membrane curvature, coaxing membrane into predestinedtube. The internal stress exerted by the fibrous components of theconnective membrane could be an essential contributor to membranecurvature and tube formation. The size of the nascent tube variesdepending on the number of crescent fusions at earlier stage. However,even the pre-tube appears to exert the ability to fuse with other tubes(see, e.g., FIG. 14H). Depending on fusion site, the outcome may be atube of larger diameter, or extended length, or the formation of abranching tube (see, e.g., FIG. 11 and FIG. 14H). Physical properties ofthe tube (including but not limited to, flexibility, ability to fuse,ability to branch, etc.) may relate to membrane chemical constitution,which is suggested by the observed different susceptibility of nascenttubes to different dyes.

Membrane fenestration: Some of the crescent formations merge to createfenestrations (holes) in the connective membrane. These may appear likecircular windows of various diameters opening to other layers (see,e.g., FIG. 14I and FIG. 14J). The white background observed in thesefigures is mostly crammed with unstained, almost unrecognizable, SMScells. These fenestrations provide access for fluids (includingnutrients or signal molecules) into other histological layers. Sincemany tubes of the present mesh assembly appear well connected to thesefenestrations (see, e.g., FIG. 14I), they may also permit easy access offluids to the tubular mesh assembly system. In addition, they mayprovide depending on diameter easy access for individual cells, cellaggregates and/or cell constructs.

Comparison of observed structures with sheep heart tissue: The structurein FIG. 15A is consistent with the one observed in three dimensionalcluster mesh assembly in cell culture (see, e.g., FIG. 9H). In FIG. 15B,the structure is similar to intermediary step of the coated tubularassembly. Two dimensional leaf mesh assembly stained using Amido blackis observed in FIG. 15C and related structures stained with MassonGoldner trichrome in FIG. 15D. Clearly observed in stained preparationare also: crescent formation (see, e.g., FIG. 15E) Membrane Tubetransition (see, e.g., FIG. 15F, FIG. 15G). Fenestrations are alsoobvious (see, e.g., FIG. 15H) but may take at times different shapesthan the one produced in SMS cell culture (see, e.g., FIG. 15I).

Example 6 Screening Sheep/Rat Tissues For Cells With Characteristic SMSCell Shape

Screening various tissues from sheep and rat organs indicated theabundant presence of cells that have the exceptional characteristicshape of SMS cells (see, FIGS. 17 to 39). Microscopic examination usinghistochemical staining demonstrate also the presence of the tissue likestructure observed in the in Vitro SMS cell culture (see, FIG. 19, FIG.21, FIG. 34). Several obtained microscopic pictures of various stainedtissues suggest farther a continuum in the process of differentiation,from the small simple SMS cell type to other type differentiated cells(see, FIG. 22, FIG. 24, FIG. 34). This is suggested by a gradual shift,from the typical characteristic morphology of SMS cells, to otherdiverse cellular forms (see, FIG. 19, FIG. 26, FIG. 31, FIG. 39).

Example 7 Neuronal Antigens in Differentiated SMS Cells

Cells obtained by in vitro neurogenic differentiation of cultured SMScells were tested for various antigens that are typical to neuronalcells. Testing using antigen specific fluorescent antibodies resulted inpositive signals for the three examined neuronal antigens (class IIIbeta Tubulin, Glial fibrillary acidic protein GFAP, Myelin proteolipidprotein PLP) (see, FIG. 40, FIG. 41, FIG. 42).

The above description of the disclosed embodiments to enable any personskilled in the art to make or use the invention. Various modificationsto these embodiments will be readily apparent to those skilled in theart, and the generic principles described herein can be applied to otherembodiments without departing from the spirit or scope of thedisclosure. Thus, it is to be understood that the description anddrawings presented herein represent an exemplary embodiment of thedisclosure and are therefore representative of the subject matter whichis broadly contemplated by the present invention. It is furtherunderstood that the scope of the present disclosure fully encompassesother embodiments that may become obvious to those skilled in the artand that the scope of the present disclosure is accordingly not limited.

1-45. (canceled)
 46. A method of isolating small mobile stem (SMS) cellscomprising: culturing cells in media, wherein the cells are obtainedfrom umbilical cord blood, peripheral blood, bone marrow, or a solidtissue for at least 27 passages; identifying adherent cells that arefrom 4.5 to 5.5 μm in size; and isolating the identified cells, whereinthe isolated cells are SMS cells.
 47. The method of claim 46, whereinthe solid tissue is placenta, liver, heart, brain, kidney or gastrointestinal tract.
 48. The method of claim 46, wherein the media is DMEMlow glucose or DMEA high glucose, optionally with Hepes or serum. 49.The method of claim 46, wherein the cultured cells are passaged uponreaching 80% to 90% confluency.
 50. The method of claim 46, wherein thecultured cells are passaged using a scraper or cold shock treatment. 51.The method of claim 46, further comprising producing a protein from saidisolated SMS cells.
 52. The method of claim 46, further comprisingproducing a compound from said isolated SMS cells.
 53. The method ofclaim 46, further comprising using said isolated SMS cells for anindustrial application.
 54. A method for testing the safety or efficacyof a drug comprising: exposing an isolated population of small mobilestem (SMS) cells to a drug; and evaluating the safety or efficacy of thedrug.
 55. The method of claim 54, wherein the isolated population of SMScells are obtained by: culturing cells in media, wherein the cells areobtained from umbilical cord blood, peripheral blood, bone marrow, or asolid tissue for at least 27 passages; identifying adherent cells thatare from 4.5 to 5.5 μm in size; and isolating the identified cells,wherein the isolated cells are SMS cells.
 56. The method of claim 55,wherein the media is DMEM low glucose or DMEA high glucose, optionallywith Hepes or serum.
 57. The method of claim 54, wherein the SMS cellsoriginate from umbilical cord blood, peripheral blood, bone marrow, or asolid tissue.
 58. The method of claim 57, wherein the solid tissue isplacenta, liver, heart, brain, kidney or gastro intestinal tract.
 59. Amethod of producing extracellular matrix (ECM) proteins from an isolatedpopulation of small mobile stem (SMS) cells, the method comprisingproducing ECM proteins from a population of isolated SMS cells in a cellculture.
 60. The method of claim 59, wherein the population of isolatedSMS cells are obtained by: culturing cells obtained from umbilical cordblood, peripheral blood, bone marrow, or a solid tissue for at least 27passages; identifying adherent cells that are from 4.5 to 5.5 μm insize; and isolating the identified cells, wherein the isolated cells areSMS cells.
 61. The method of claim 60, wherein the solid tissue isplacenta, liver, heart, brain, kidney or gastro intestinal tract. 62.The method of claim 59, further comprising incubating the SMS cells at37° C. and 5% CO₂ in a humidified atmosphere.
 63. The method of claim59, wherein the ECM proteins are organized as layers of membranes.